Evaluation of the Protoscolicidal Activity of Humulus lupulus Methanolic Extracts on Echinococcus granulosus Sensu Stricto

The larval stage of the parasite Echinococcus granulosus sensu lato (s.l) is responsible for cystic echinococcosis (CE), a long-term infection affecting humans and animals worldwide, and constitutes a serious public health concern. If left untreated, CE can cause serious damage to multiple organs, especially the liver and lungs. Regarding the treatment, in the last few years, the use of pharmacological treatment has increased, suggesting that in the future, drug therapy may replace surgery for uncomplicated cysts. However, the only available anthelmintic drug to treat this infection is the albendazole, which has an efficacy that does not exceed 50%. On the basis of the above-mentioned evidence, new and improved alternative treatments are urgently needed. The use of natural products and their active fractions and components holds great promise as a valuable resource for the development of novel and effective therapies. Hop (Humulus lupulus L.) is a bittering agent in the brewing industry for which the sedative, digestive, anti-inflammatory, and antimicrobial effects have been reported. The purpose of this study was to assess the in vitro efficacy of methanolic extracts from the leaves of hop varieties against E. granulosus sensu stricto (s.s) protoscoleces. Varieties Mapuche and Victoria caused a stronger protoscolicidal effect compared to the Bullion, Cascade, and Traful varieties (P < 0.01), coinciding with their highest content of flavonoids, total polyphenols, and saponins. The viability of protoscoleces treated with the varieties Mapuche and Victoria decreased to approximately 50% at days 5 y 8, respectively, showing alterations such as soma contraction and impaired microtriches. After 18 days of treatment with both varieties, protoscoleces were completely altered both structurally and ultrastructurally. In conclusion, the methanolic extracts of the H. lupulus varieties Mapuche and Victoria demonstrated a marked in vitro effect against E. granulosus s.s. protoscoleces. The beer-making industry exclusively uses hop cones, leaving behind large amounts of hop leaves as an agricultural by-product that is not being utilized. On the basis of our study, we propose that hop leaves could also be used as a source of secondary metabolites with anthelmintic activity.


Introduction
Te larval stage of the parasite Echinococcus granulosus sensu lato (s.l) is responsible for cystic echinococcosis (CE), a long-term infection afecting humans and animals worldwide, and constitutes a serious public health concern [1].
Te life cycle of E. granulosus s.l. is complex and involves an intermediate host (wild or livestock mammals and occasionally humans) and a defnitive host (usually dogs and other canids) which contain the adult worm in the intestine and release the eggs along with the feces.Eggs may be accidentally ingested by an intermediary host and develop a hydatid cyst flled with hydatid fuid and protoscoleces [2].
Tis parasite can cause serious damage to multiple organs, especially the liver and lungs, which can lead to death if left untreated in time.Based on the type of cysts, strategies for treating CE include surgery, percutaneous management, chemotherapy with benzimidazoles, and, for inactive cysts, a "watch and wait" approach [3].Over the last few years, the use of pharmacological treatment has increased, suggesting that in the future, drug therapy may replace surgery for uncomplicated cysts [4].
Albendazole (ABZ), a benzimidazole derivative, is the primary anthelminthic drug used to treat this infection.However, its liposoluble nature and poor bioavailability contribute to its efcacy not exceeding 50% in treating CE [3].Consequently, there is a critical need for the development of new drugs that can overcome these drawbacks and provide improved therapeutic outcomes for patients with this infection.
In this scenario, the use of natural products and their active fractions and components holds great promise as a valuable resource for the development of novel and effective therapies.Tey exhibit powerful pharmacological activities such as anti-infammatory, antioxidant, or antiproliferative efects and have been proven to be accessible, relatively low in cost, and have generally low toxicities [5].
In recent years, there has been a surge in the investigation of various plant extracts as potential therapeutic agents against CE, aiming to identify alternative natural compounds for efective treatment.Tese studies have yielded numerous encouraging outcomes, highlighting the potential of plant-derived substances in combating CE [6][7][8][9].
Hop (Humulus lupulus L.) is primarily known as a bittering agent in the brewing industry [10].However, its uses have diversifed in recent times.It is valued in phytotherapy because of its anti-infammatory, digestive, sedative, and antimicrobial properties [11].Also the antiparasitic activity was shown for some hoppurifed compounds on Trypanosoma brucei [12].
Te purpose of this study was to assess the in vitro effcacy of methanolic extracts from leaves of hop varieties against E. granulosus sensu stricto (s.s) protoscoleces.

Preparation of H. lupulus Methanolic Extract.
Te extraction of secondary metabolites coming from the diferent varieties was carried out by using the mixture of methanolwater (50 : 50).Te extraction was performed by placing 1 gram of leaves (in triplicate) in 30 mL of the solvent for 3 h in an ultrasound bath at 40 °C.Ten, the suspension was centrifuged for 10 min at 8000 rpm.Te obtained supernatant was placed in falcon tubes and conserved at 4 °C in dark until use [13].

Chemical Characterization of H. lupulus Methanolic Extracts
2.3.1.Quantifcation of Total Polyphenol Content.Te total content of phenolic compounds in the diferent extracts was determined by the Folin-Ciocalteu method, according to the procedure reported by Singleton and Rossi [14] with some modifcations.A calibration curve was made using 0, 2, 6, 8, 10, 15, and 20 μg•ml −1 of a standard solution of gallic acid (GA) (200 μg•mL −1 ) and the total polyphenol content of each extract was estimated from the linear regression obtained.
Te absorbance was measured at 760 nm, and the total polyphenol content was calculated in mg gallic acid equivalents (GAE) per g of dry extract.

Quantifcation of Total Flavonoid Content. Woisky
and Salatino [15] methods with some modifcations was used for the determination of total favonoid content.Briefy, a calibration curve was constructed using 0, 2, 4, 6, 10, 14, 18, and 22 μg•mL −1 from a quercetin (QE) standard solution (200 μg•mL −1 ) and the total favonoid content of each extract was determined from the linear regression obtained.Te absorbance was measured at 420 nm using an Agilent 8453 UV-visible spectrophotometer with a diode array, and the total favonoid content was calculated as mg QE equivalents per g of dry extract.

Quantifcation of Total Saponin
Content.Te determination of total saponin content (TSC) was performed according to Le et al. [16], with minor changes.Te calibration curve was constructed with oleanoic acid (OA) as a reference standard, and concentrations between 0.001 and 0.005 μg•mL −1 of OA were used for the calibration curve.An Agilent 8453 UV-visible spectrophotometer with a diode matrix was used to measure absorbance at 560 nm, and the data was recorded.Te TSC of each extract was obtained from the linear regression of the calibration curve and calculated as mg AO equivalent per g of dry extract.For all the determinations, measurements were carried out in quadruplicates.

Parasite Material and Protoscoleces Collection.
Hepatic and pulmonary hydatid cysts were obtained from cattle slaughtered in an abattoir located in the province of Buenos Aires, Argentina.Protoscoleces were removed aseptically from cysts and washed several times with phosphate-bufered saline (PBS, pH 7.2).Viability was 2 Evidence-Based Complementary and Alternative Medicine assessed by the methylene blue exclusion test [17].Parasitic material was genotyped by sequencing a fragment of the gene coding for mitochondrial cytochrome c oxidase subunit 1 (CO1), as previously described [18].Te G1 genotype was identifed through sequencing analysis.
2.5.In Vitro Protoscolicidal Activity.Viable and free protoscoleces (2000 per Leighton tube) were cultured in 6 mL of culture medium 199 at 37 °C with no change of medium throughout the experiment as previously described [17].Te extracts of the diferent H. lupulus varieties (Bullion, Cascade, Traful, Mapuche, and Victoria) were added at the fnal concentration of 100 µg/mL.Control protoscoleces were incubated in culture medium or in culture medium with methanol (25 µL/mL).Cultures were performed in triplicate, and each experiment was repeated three times, each time with a diferent batch of protoscoleces.To determine the appearance of morphological changes, culture tubes were monitored under a microscope daily.Viability assessment using the methylene blue exclusion test was performed every day until day 4 and then, every 3 days.Samples of protoscoleces from each experimental group were periodically taken for ultrastructural studies using scanning electron microscopy (SEM).
2.6.Electron Microscopy.Samples of protoscoleces collected in the in vitro studies were processed for SEM following the protocol described by Elissondo et al. [19].

Statistical Analysis.
All statistical analyses were conducted within the R environment [20].P values less than 0.05 were regarded as statistically signifcant.For the in vitro incubation of protoscoleces with the extracts of the diferent H. lupulus varieties, a generalized linear model (GLM) with a binomial distribution of the error was ftted with the proportion of viability as a response variable and treatments and time in days as explanatory variables.In order to determine if time-treatment interactions should be included in the model, we used the "ANOVA" command from the "car" package [21].Diferences among the H. lupulus varieties and control were assessed by pairwise contrasts of the interaction means using the "emmeans" package [22].Protoscoleces viability is reported as the predicted value of the model with 95% confdence in the upper and lower limits.Te study avoided any unnecessary animal sufering.Animals were housed in a temperature-controlled (22 ± 1 °C), light-cycled (12 h light/dark cycle) room and received food and water ad libitum.

Results
Chemical characterization of the diferent H. lupulus varieties is shown in Table 1.Te varieties Mapuche and Victoria presented the highest content of favonoids, total polyphenols, and saponins.Te values are showed, as the average of analyses conducted with quadrupled ± standard deviation.
Te survival of E. granulosus s.s.protoscoleces incubated with diferent H. lupulus varieties is shown in Figure 1.Varieties Mapuche and Victoria had a stronger efect than varieties Bullion, Cascade, and Traful (P < 0.01).Te viability of protoscoleces treated with the varieties Mapuche and Victoria decreased to approximately 50% at days 5 y 8, respectively, whereas the treatment with the varieties Traful and Cascade caused a decrease in the viability of 65% at the end of the experiment.Te treatment with the variety Bullion did not show any diference with the control (P � 0.71).
Te results of the viability test coincided with the tegumental alterations observed daily by an inverted microscope and with the tissue damage determined at the ultrastructural level.Protoscoleces treated with the variety Mapuche showed soma contraction, rostellar disorganization, and presence of blebs in the tegument after 3 days of treatment (Figure 2(b)) and the presence of severe impairment leading to death after 9 days of treatment (Figure 2(d)).Te treatment with the variety Victoria caused the frst morphological alterations at day 6 (Figure 2(c)) becoming more pronounced at day 12 (Figure 2

Discussion
Based on the inadequate therapeutic options available for numerous parasitic infections impacting humans and animals, coupled with the emergence of drug resistance, the utilization of medicinal plants has gained signifcance as a potential reservoir for the discovery of anti-parasitic drugs [23].
To fnd new active drugs as potential treatment options for CE, we used in vitro cultured E. granulosus s.s.protoscoleces to study the activity of diferent varieties of H. lupulus extracts for the frst time.
Te application of hop as a medicinal plant has more than 2000 years of history [24].Among their interesting Evidence-Based Complementary and Alternative Medicine biological properties are their antiviral, antibacterial, antifungal, anti-infammatory, and anticancerogenic activities.
Its strong pharmacological activity and potential therapeutic application are due to the presence of a wide range of bioactive molecules, mainly secondary metabolites which confer particular features to the plant [25].
In the last decades, several phytochemical studies have been conducted to examine the composition of hop cones and other parts of the plant, resulting in the isolation and identifcation of pharmacologically relevant compounds based on increasing interest in the health benefts of this plant [26,27].
Over time, breweries have developed distinct varieties of H. lupulus, leading to extracts with diferent properties that may contribute to combating diseases [28].
Te secondary metabolites saponins and polyphenols play a crucial role in the plant's defense mechanisms against external biotic and abiotic factors [29].
Te composition and amount of secondary metabolites present in hops are mainly dependent on the variety, which is linked to its genetic potential to synthesize certain substances [30].
Te varieties Victoria, Mapuche, Cascade, Traful, and Bullion were included in our study.Varieties Mapuche and   Evidence-Based Complementary and Alternative Medicine Victoria showed the highest anthelmintic efect.Interestingly, the variety Mapuche had the highest content of total phenolic compounds (P < 0.05), favonoids (P < 0.01), and saponins (P < 0.01).
Structural and ultra-structural changes observed by light microscopy and SEM were similar to those reported in previous investigations that studied the anthelmintic efect of medicinal plant extracts [7,8,31] or isolated compounds such as carvacrol, thymol, and beta-myrcene against E. granulosus s.s protoscoleces [32][33][34].
In particular, a recent study assessed the hop varieties Victoria and Mapuche against Varroa destructor, a parasitic bee mite.Both varieties exhibited signifcant toxicity against mites; nevertheless, Victoria proved to be the most potent, highlighting its potential as a promising compound for acaricidal treatment [35].Te diferences observed in our study between the assessed hop varieties could be attributed to the varying content of secondary metabolites observed in each.It has been stated that saponin compounds, combined with the synergy produced by other components, have a high potential to break cells, hence increasing the cell death process [36].On the other hand, antimicrobial activity of hop polyphenols was demonstrated.Te antimicrobial mechanism involves the interaction with phospholipids of the cell membrane, leading to destabilization, leakage of cellular contents, and ultimately cell death [37].

Conclusion
Te methanolic extracts of the H. lupulus varieties Mapuche and Victoria proved to have a signifcant in vitro efect on E. granulosus s.s.protoscoleces.Te beer-making industry exclusively uses hop cones, leaving behind large amounts of hop leaves as an agricultural by-product that is not being utilized.On the basis of our study, we propose that hop leaves could also be used as a source of secondary metabolites with anthelmintic activity.

Figure 1 :
Figure 1: Survival of E. granulosus s.s.protoscoleces after in vitro exposure to 100 µg/ml of the diferent Humulus lupulus varieties.Te ribbons and lines show the 95% confdence intervals and the predicted fts from a generalized linear mixed-efects model.

Figure 3 :
Figure 3: Scanning electron microscopy of E. granulosus s.s.protoscoleces incubated in vitro with the diferent Humulus lupulus varieties.Control protoscoleces show the characteristic structure: soma (s), suckers (su) and rostellum (r) (a, b).Protoscoleces treated during 3 days with the variety Mapuche (c) and during 6 days with Victoria (d) showed soma contraction and altered microtriches.After 18 days of treatment with both varieties, protoscoleces were completely altered (e, f ).

Table 1 :
Content of total phenolic compounds, total favonoids and total saponins of the diferent Humulus lupulus varieties extracts.